A comparison of anther and microspore culture as a breeding tool in Brassica napus

Summary A direct comparison of microspore culture and anther culture was made in Brassica napus using F₁ crosses of Regent (canola) by Golden (rapeseed), and their reciprocals, as well as a hybrid between Reston and a highly embryogenic, canola-quality breeding line (G231) as donor plants. The study confirmed that microspore culture can be ten times more efficient than anther culture for embryo production. Embryo yields from cultures initiated from the Reston x G231 were four-fold greater than those initiated from the Regent x Golden crosses, and significant differences were also detected among cultures initiated from the different Regent x Golden crosses. These results illustrate the influence that donor plant genotype has on embryo production. However, superior embryogenic potential among donor material was not always coincident with superior plant production. The average haploid-todiploid ratio in microspore-derived regenerates was 21 for the population obtained from the Regent x Golden crosses but 11 for the Reston x G231 cross. For both types of material, the frequency of diploids increased upon repeated cycles of explanting. A field study showed that there were no differences between the populations of anther-derived and microspore-derived spontaneous diploid and doubled haploid lines, with respect to the days required for them to flower or to mature. The information is valuable for canola breeding programs considering the use of haploidy.     

Genetics of self incompatibility in diploid Ageratum houstonianum mill

Summary Diallels and backcrosses among self-incompatible (SI) clones and progeny of Ageratum houstonianum Mill. could be organized into intra-incompatible classes. Four of 5 progenies segregated in expected ratios of S genotypes. Ageratum expressed a one-locus incompatibility system of the sporophytic type with a linear dominance series of multiple alleles and complete allelic dominance in both pollen and stigma. In the second part of the study, a high percentage of self-seed set was observed during the first flowering of a progeny from a pseudo-self compatible (PSC) seed source. Six progenies were derived from the PSC seed source. Five of the 6 segregated PSC SI plants, 4 of which fit a 3 1 ratio of PSC SI plants. All plants of the sixth progeny were SI. Two F₁ progenies with the same PSC pollen parent produced significantly different segregations of PSC SI plants. It appeared that PSC acted as a major gene when the most recessive S allele was also present, but PSC was not expressed when the most dominant S allele was present. Clones propagated from PSC plants were SI and cross incompatible with a related S-allele tester. Thus, PSC was transient in that it was apparent in seed-propagated plants but not in plants clonally propagated from the PSC individuals.Scientific Journal Series Paper Number 12,299 of the Minnesota Agricultural Experiment Station     

Sex determination in the dioecious Melandrium

Summary Melandrium album (2n=24), a dioecious species with heteromorphic sex chromosomes (XY, males and XX, females), has a strong genetic commitment for sex determination. We report here a procedure for obtaining haploid plants from cultured anthers and show that genotype, pollen stage, cold treatment and certain culture media components are essential for a reproducible yield of embryos. Our procedure increased the number of responsive anthers and not the number of responsive microspores per anther. Most likely, our experimental system allows the recovery of competent microspores, and this on a medium containing either an auxin or a cytokinin. All of the 36 anther-derived plants tested expressed a female phenotypic sex instead of the theoretical one male one female ratio. When analysed cytologically, the plants exhibited the corresponding female genetic sex (one or two X chromosomes).     

Morphological development of anthers induced by the dimorphic smut fungus<Emphasis Type="Italic"> Microbotryum violaceum</Emphasis> in female flowers of the dioecious plant<Emphasis Type="Italic"> Silene latifolia</Emphasis>

When inoculated with the dimorphic smut fungus Microbotryum violaceum (Pers.) G. Deml and Oberwinkler, the female flower of the dioecious plant Silene latifolia (Miller) E.H.L. Krause develops anther-like structures filled with spores instead of pollen grains. Using natural scanning electron microscopy, Nomarski interference microscopy, and fluorescence microscopy, we investigated the morphological modifications of the host plant resulting from this parasitism and the localization of smut hyphae in the flower bud. Flowers of infected plants lasted significantly longer than those of healthy plants, probably because the infection strengthened floral organs, such as the flower base and the anther filaments. Smut hyphae were observed throughout all organs of the young flower buds of infected plants, including sepals, petals, stamens, and pistil primordia. In healthy female flowers, anthers initiated sporogenous cell formation, but lacked parietal cell layers. By contrast, the parietal cell layers of infected female flowers differentiated into tapetal tissue, middle cell layers, and endothecial layers, as in the anthers of healthy male flowers. Smut spore formation in the infected anther was initiated in intercellular regions between the sporogenous cells, resulting in degeneration of premature sporogenous cells, tapetal tissue, and middle cell layers. The development of the endothecial layers and epidermis in the infected anther were morphologically normal.Abbreviations DAPI 4,6-diamidino-2-phenylidole - i infected - PMC pollen mother cell     

Production of androgenic dihaploid lines of the disomic tetraploid potato species Solanum acaule ssp. acaule

Over 250 dihaploid lines derived from a disomic tetraploid genotype of Solanum acaule ssp. acaule Bitt. (acc. PI 472655) were produced via androgenesis. The anther donor plant had previously shown immunity to bacterial ring rot caused by Clavibacter michiganensis ssp. sepedonicus (Spieck. and Kotth.) Davis et al., and has now been shown to have high embryogenic capacity in anther culture. In total, 370 shoots were regenerated from 4,011 anthers cultured. The ploidy level of the 287 regenerants was determined from greenhouse-grown plants using flow cytometry. Of these plants, 274 (95%) were dihaploids with an average DNA content of 1.68 pg, approximately half that of the tetraploid anther donor (2.95 pg). The remainder of the anther-derived regenerants (5%) were tetraploid, hexaploid or mixoploid. Chromosome counts confirmed the results obtained by flow cytometry. In the greenhouse, none of the 33 dihaploid lines analysed produced berries but showed low (2%) male fertility. This contrasted with five greenhouse-grown tetraploid anther-derived plants which produced berries and seeds. Comparison of the general leaf morphology and floral characteristics of the tetraploid anther donor, S. acaule, and the dihaploids indicated that little variation exists in this species. Received: 28 August 1997 / Revision received: 22 December 1997 / Accepted: 27 July 1998     

Morphological patterns in Petunia hybrida plants regenerated from tissue cultures and differing by their ploidy

Summary Quantitative variation in seven morphological characteristics (leaf length and width, leaf length/ width ratio, flower, petal and stomata length, and number of chloroplasts in guard cells) were studied in Petunia hybrida plants regenerated from anther tissue culture and belonging to four different classes of ploidy (2n, 2n–3n, 3n–2n, 4n–8n). Results showed that leaf size is not a good characteristic for discriminating between plants of different ploidy — flower and stomata characteristics being more adequate for this purpose. After applying stepwise discriminant analysis the association chloroplast number — leaf length/width ratio — petal length was verified to be more appropriate for the discrimination of ploidy classes.     

The influence of anther cold pretreatments and donor plant genotypes on in vitro androgenesis in wheat (Triticum aestivum L.)

Cold pretreatments applied to excised anthers in liquid potato 2 medium proved to be unnecessary. Generally, cold pretreatments inhibited anther response and productivity as the duration was lengthened or as the pretreatment temperature was lowered. There were significant differences in response attributed to the anther donor genotype. Green and albino plants as well as roots only have been regenerated from the spring wheat cultivars Sinton, Neepawa, Pitic 62 and DW 50. Most plants were haploid.     

Diploid somatic-hybrid plants regenerated from rice cultivars

Summary Somatic hybrid plants were obtained between rice cultivars Yamahoushi and Murasakidaikoku. Since Murasakidaikoku is a double mutant having both dominant (purple coloration) and recessive (dwarf) markers, the somatic hybrids can be easily distinguished from their parents. Protoplasts were isolated from anther-derived calli, and electrofused protoplasts were cultured without selection of hybrid cells. Out of 27 regenerated plants, 9 proved to be hybrids based on their purple coloration and normal plant type, traits which were identical to those of the sexual F₁ hybrid between the same parental cultivars. The somatic hybrids included three diploid and six triploid plants. Segregation of parental markers was observed in the selfed progenies. These results demonstrated that diploid hybrids of rice could be obtained through somatic hybridization between haploid anther-derived cells instead of by sexual hybridization.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid     

Ribulose Bisphosphate Carboxylase Activity in Anther-Derived Plants of Saintpaulia ionantha Wendl. Shag

Plants obtained from anther culture of the African violet, Saintpaulia ionantha Wendl. `Shag' and vegetatively cloned copies of the parent anther donor plant were examined for their ploidy and ribulose-1,5-biphosphate carboxylase (RuBPcase) activity. The cloned parent plants were all diploid and did not vary much in their nuclear DNA, chlorophyll, and RuBPcase activity. Some of the anther-derived plants were similar to the parent plants while others were not. Different levels of ploidy were observed among the androgenetic plants. RuBPcase activities higher than that of the parent plants were found in some anther-derived plants. However, there was no direct correlation between ploidy and RuBPcase activity. Expression of nuclear genes from a single parent in the anther-derived plants and it's diploidization or plastid changes during early stages of microsporogenesis or androgenesis are suggested as possible reasons for the variations observed among them. This could be a useful technique to obtain physiological variants which could be agronomically desirable.     

Targeted mapping and linkage analysis of morphological isozyme,and RAPD markers in peach

Nine different F2 families of peach [Prunus persica (L.) Batsch] were analyzed for linkage relationships between 14 morphological and two isozyme loci. Linkage was detected between weeping (We) and white flower (W), 33 cM; double flower (Dl) and pillar (Br), 10 cM; and flesh color (Y) and malate dehydrogenase (Mdh1), 26 cM. A leaf variant phenotypically distinct from the previously reported wavy-leaf (Wa) mutant in peach was found in progeny of Davie II. The new willow-leaf character (designated Wa2) was closely linked (0.4 cM) to a new dwarf phenotype (designated Dw3). Two families derived from the pollen-fertile cultivar White Glory segregated for pollen sterility, but segregation did not follow a 31 ratio. Evidence is presented suggesting that White Glory possesses a pollen-sterility gene (designated Ps2) that is non-allelic to the previously reported pollen-sterility gene (Ps) in peach. Ps2 was linked to both weeping (We-Ps2, 15.5 cM) and white flower (Ps2-W, 25.3 cM). A genomic map of peach containing 83 RAPD, one isozyme, and four morphological markers was generated using an F2 family obtained by selfing an NC174RL x Pillar F1. A total of 83 RAPD markers were assigned to 15 linkage groups. Various RAPD markers were linked to morphological traits. Bulked segregant analysis was used to identify RAPD markers flanking the red-leaf (Gr) and Mdh1 loci in the NC174RL x Pillar and Marsun x White Glory F2 families, respectively. Three markers flanking Mdh1 and ten markers flanking Gr were identified. The combination of RAPD markers and bulked segregant analysis provides an efficient method of identifying markers flanking traits of interest. Markers linked to traits that can only be scored late in development are potentially useful for marker-aided selection in trees. Alternatives for obtaining additional map order information for repulsion-phase markers in large F2 populations are proposed.This work was supported in part by the McKnight Foundation, North Carolina Biotechnology Center, North Carolina State University Forest Biotechnology Research Consortium, and the North Carolina Agricultural Research Service, Raleigh, North Carolina     

Variation of RuBP carboxylase activity among anther-derived plants of Solanum phureja

The effect of genotype and ploidy on RuBP carboxylase (EC 4.1.1.39) activity, chlorophyll content, leaf area, chloroplast ultrastructure and not photosynthesis among monoploid. diploid and tetraploid anther-derived plants of Solanum phureja Juz, and Buk. was studied. Within the monoploid group, RuBP carboxylase activity and concentration displayed a significant genotypic effect. For the diploids, variation among genotypes was significant for total protein content and maximum specific activity of RuBP carboxylase, and among the tetraploids for net photosynthesis and specific leaf weight. Ploidy effect was evident regarding net photosynthesis, leaf area and chlorophyll content. The different ploidy groups among the anther-derived plants surpassed the anther donor plant for all characteristics except maximum activity of RuBP carboxylase and net photosynthesis. For the latter only the tetra-ploid group was superior to the anther source plant. However, a monoploid genotype with an increase of 9% in maximum activity of RuBP carboxylase over the anther-donor plant was identified. Segregation of trails rind differential gene expression together with possible mutations during androgenesis are discussed as sources of variation.     

CLUSTER ANALYSIS OF ANTHER-DERIVED PLANTS OF SOLANUM PHUREJA (SOLANACEAE) BASED ON MORPHOLOGICAL CHARACTERISTICS

The feasibility of grouping anther-derived plants of Solarium phureja according to ploidy based on their morphological characteristics was studied. Canonical discriminant analysis identified four characteristics (anther length, number of chloroplasts per pair of guard cells, leaf width, corolla width at widest diam) of nine measured as the most effective combination for diagnosing ploidy. Data for these characteristics from two sets of plants were subjected to two clustering techniques, one using the average linkage clustering (UPGMA of the NT-SYS programs) and the other using centroid sorting (SAS-Fastclus). Screening of anther-derived plants by cluster analysis proved to be an efficient means of separating monoploids from the other ploidy levels.     

Identification of two RAPD markers tightly linked with the Nicotiana debneyi gene for resistance to black root rot of tobacco

Linkage of randomly amplified polymorphic DNA (RAPD) markers with a single dominant gene for resistance to black root rot (Chalara elegans Nag Raj and Kendrick; Syn. Thielaviopsis basicola [Berk. and Broome] Ferraris) of tobacco (Nicotiana tabacum L.), which was transferred from N. debneyi Domin, was investigated in this study. There were 2594 repeatable RAPD fragments generated by 441 primers on DNAs of Delgold tobacco, a BC₅F₈ near isogenic line (NIL) carrying the resistance gene in a Delgold background, and PB19, the donor parent of the resistance gene. Only 7 of these primers produced eight RAPD markers polymorphic between Delgold and PB19, indicating there are few RAPD polymorphisms between them despite relatively dissimilar pedigrees. Five of the eight RAPD markers were not polymorphic between Delgold and the NIL. All of these markers proved to be unlinked with the resistance gene in F₂ linkage tests. Of the remaining three RAPD markers polymorphic between Delgold and the NIL, two were shown to be strongly linked with the resistance gene; one in coupling and the other in repulsion. Application of the two RAPDs in the elimination of linkage drag associated with the N. debneyi resistance gene and marker-assisted selection for the breeding of new tobacco cultivars with the resistance gene is discussed.     

Variation for improved protein and yield from rice anther culture

Summary The anther culture technique is useful for the recovery of haploids which when doubled provide homozygosity. Additionally, beneficial as well as deleterious genetic and epigenetic changes are promoted by the in vitro procedures. The majority of plants recovered from anther calli of the cultivar Calrose 76 were similar to the starting cultivar but plants regenerated from microspore calli had a wider range of responses than controls for several characteristics. Plants with larger seeds, higher levels of seed protein, shorter stature and more highly tillered than the starting cultivar were obtained from selfed anther-derived plants. The data also support the concept that in vitro procedures including anther culture of specific cultivars of rice promoted the recovery of phenotypes with increased seed storage proteins.Contribution from the U.S. Department of Agriculture, Science and Education, Agricultural Research Service, Plant Physiology Institute, Tissue Culture and Molecular Genetics Laboratory, Room 116, Building 011-A, BARC-West, Beltsville, Maryland 20705, USA     

Variation in leaf carbon isotope discrimination in Encelia farinosa: implications for growth,competition, and drought survival

Population-level variation in the leaf carbon isotope discrimination () values was examined in Encelia farinosa, a common Sonoran Desert shrub. There was approximately a 2 range in values among different plants. These differences in values among neighboring plants were maintained through time, both under conditions when neighbors were present and after neighbors had been removed. Individuals with high values were found to have an accelerated growth rate when these plants were released from competition for water. Individuals with low values were better able to persist through long-term drought. These data suggest possible tradeoffs between conditions favoring high- and low--value plants within a natural population. Given the temporal variability in precipitation between years and spatial variability in microhabitat quality in the Sonoran Desert, variation in values among E. farinosa plants will be maintained within a population.     

Promoter sequences from two different Brassica napus tapetal oleosin-like genes direct tapetal expression of β-glucuronidase in transgenic Brassica plants

To investigate the sequences responsible for the regulated expression of tapetal-specific oleosin-like genes, ca. 2 kb of the 5-upstream regions from two divergent genes, OlnB;4 and OlnB;13, were isolated, sequenced and fused to the reporter gene -glucuronidase for study in transgenic Brassica napus plants. Although the proteins encoded by these two genes are highly divergent, except for the conserved oleosin-like domain, the first 250 bp of their 5-upstream regions was 86% identical, including a region of 150 bp upstream from the TATA box. Analysis of 42 independent transformants by histochemical and fluorometric methods showed that both promoters directed tapetal-specific expression that peaked at the 4 mm flower bud stage.     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Glycoconjugates of the intestinal epithelium of the domestic fowl (Gallus domesticus): a lectin histochemistry study

Summary A lectin histochemical study was performed on formalin-fixed paraffin-embedded tissues of duodenum, jejunum, ileum, caecum and colon from six fasted and six non-fasted 8-week-old chickens (Gallus domesticus). The purpose of this study was to identifyin situ the pattern of carbohydrate residues present on the luminal surface of the intestinal epithelium. Ten biotinylated lectins with different sugar specificities were used as probes, and avidin—biotin—peroxidase complex (ABC) was used as a visualant. The most significant finding was the binding pattern ofLens culinaris agglutinin to various segments of the intestines. The luminal surface of the small intestinal epithelium did not stain with this lectin. In the colon the luminal surface was lightly stained, while the caecal luminal surface was intensely stained. Throughout the intestine the luminal surface stained withCanavalia ensiformis agglutinin,Ricinus communis agglutinin-I and wheatgerm agglutinin, but it did not stain withDolichos biflorus agglutinin. These findings indicated that, throughout the intestine, the luminal surface contains glycoconjugates with eitherN- orO-linked glycoprotein, or both, with terminal non-reducing -galactosyl and sialyl residues. Furthermore, the caecal surface is rich inN-linked glycoproteins with an -(16)-linked fucosyl residue near the glycosidic linkage. The potential significance of these observations and the role of glycoconjugates in host—parasite interaction (i.e.Eimeria sp. versusGallus domesticus) are discussed.     

Efficient in vitro regeneration of leek (Allium ampeloprasum L.) via flower stalk segments

Summary A new simple, efficient and rapid in vitro method for mass clonal propagation of leek (Allium ampeloprasum L.) plants, using small (5 mm) flower stalk (peduncle) explants, was established. Adventitious shoots were produced from single subepidermal cells. A wide variation in the percentage of regenerating explants and number of regenerated shoots per explant between individual plants within one cultivar was observed. The concentration of the growth regulators 6-benzylaminopurine and -naphthalene-acetic acid influenced the percentage of regenerating explants and the average number of regenerated shoots per explant. A combination of 10 mg.l^–1 6-benzylaminopurine and 10 mg.l^–1 -naphthalene-acetic acid, resulted in a maximum percentage of regenerating explants and a high average number of regenerated shoots per explant. The percentage of regenerating explants and the average number of regenerated shoots per explant decreased with increasing flower stalk length (age). The basal explants gave both the highest percentage of regenerating explants and average number of regenerated shoots per explant. An average of 300 shoots per flower stalk was obtained for all plants, making this new in vitro method a powerful tool in hybrid leek breeding.     

Variation in structural gender in the hermaphrodite Helleborus foetidus (Ranunculaceae): within- and among-population patterns

In hermaphrodite plants, variations in structural gender (defined as the ratio between male and female gametes) may occur at different levels (among flowers, plants, and populations). In this study, we investigated variation in four traits influencing structural gender (number of carpels, ovules per carpel, stamens, and pollen grains per stamen) within and among six distant populations of the hermaphrodite perennial herb Helleborus foetidus (Ranunculaceae) in the Iberian Peninsula. Our results show that the four traits investigated varied significantly at all levels considered. Traits influencing the female sexual component (number of carpels and ovules per carpel) showed greater variation at the lowermost levels (within flower and plant) than traits influencing the male component, which in turn varied more markedly among populations. Number of carpels per flower and number of pollen grains per anther were the most important traits affecting between-plant variation in structural gender. There was no evidence of significant plant-level trade-offs or correlations between the various male and female traits, which covaried differently across populations. The observed between-population variation in structural gender of Helleborus foetidus can be explained as a consequence of differences in self-pollination levels related to a flowers mating environment.